Novel prognostic biomarker in acute myocardial infarction

ABSTRACT

A diagnostic method for determining prognosis of a myocardial infarcted patient, wherein the amounts of FXIII protein are determined on the day of myocardial infarction (t0) and at least on the following three days (t1 to t3), wherein a lowering of FXIII amount on any one of t0 to t3 below a threshold value is indicative of an increased risk of poor prognosis.

FIELD OF THE INVENTION

The present disclosure concerns a novel prognostic biomarker in acutemyocardial infarction.

BACKGROUND ART

After acute myocardial infarction (AMI), the damaged heart tissue startsa complex series of processes aimed to repair, and eventually toreplace, the lesion by scar tissue [1].

During this period, the infarcted area is highly active biologically. Arapid turnover of cells and of structural components contributes tocreate new extracellular matrix (ECM). Preexisting ECM proteins (e.g.collagen) are digested by metalloproteinases (MMPs) and new matrix islaid down. Contextually, new cell migration and differentiationcontribute to myocardial recovery after injury [1,2].

Starting from the earliest phases of wound repair, attempted by theextensive changes of tissue architecture, the vulnerable wound isexposed to the mechanical stress of the continuous heart beating cycles.This is, besides, responsible for drastic changes of theintraventricular pressure and volume. During the healing processes,myocardial contraction cannot be avoided, so the lesion cannot ever healproperly, if compared to other tissue lesions in which the mechanicalstress is extremely lower and they can also be contained by provisionalrigid scaffold/tutors (i.e. skin lesions, bone fractures, etc).

Although all the specific reparative processes are potentiallyappropriate and well-timed, very often they fail in avoiding adverseremodelling and loss of myocardial performances due to non-optimal scarformation at the injury site or even to scar rupture.

The first step (attempt) in the reparative processes after wound occursis the formation of a three-dimensional fibrin meshwork aimed to blocklesion expansion and furnish a provisional scaffold/platform forendogenous neo-vessels formation, cell recruiting and spreading. Theearlier the process starts, the better the healing is due to prompt scarformation. In addition, proper healing steps should not be interruptedor hampered by enzymatic and/or mechanical actions (e.g. fibrin meshworkrupture due to unrestrained proteolytic activity or heart beating),specially in the earliest healing phases.

Normally, a high strength together with an extraordinary extensibilityand elasticity of the fibrin net is warranted by the action of acirculating transglutaminase, factor XIII (FXIII), which by means ofcovalent cross-bonds links fibrin chains and several other ECMcomponents changing drastically their original properties [3]. This factlets the fibrin-scaffold to work properly by counteracting lesionexpansion and furnishing the best setting and timing to have an optimalreparative process. Finally, cell migration/differentiation andneo-vessel formation are FXIII-dependent processes and stronglycontribute to myocardial healing and recovery after injury.

FXIII is a pro-enzyme of plasma transglutaminase family, consisting oftwo enzymatic A subunits (FXIII-A) and two non-catalytic B subunits(FXIII-B) [3]. It plays a critical role in generation of a stablehaemostatic plug, wound healing, tissue repair, and angiogenesis. FXIIIis present in plasma, platelets, monocytes, and macrophages, allcomponents deeply involved in infarct healing. Its key role on healingis demonstrated by the following:

(1) delayed haemorrhage in congenital FXIII deficient subjects;

(2) delayed wound healing in FXIII-deficient cases by human and animalmodels;

(3) positive effects by FXIII topical application on wound healing by invivo and in vitro studies;

(4) antiapoptotic and proangiogenic properties;

(5) effects on cell migration/differentiation into the wound;

(6) modulation of fibrin and new collagen synthesis and deposition inECM;

(7) strong positive effects in heart transplanted animals.

Extraordinary direct evidences of the essential role of FXIII in acuteand chronic infarct scar stability come from an experimental model withgenetically reduced FXIII levels in animals. Nahrendorf and coll. in2006 [4], in mice deficient in (FXIII−/−), or heterozygous for(FXIII−/+) the FXIII-A gene (FXIII levels 5% and 70% respectively), bymeans of coronary ligation and high-field cardiac MRI, followedmyocardial scar formation and the consequent remodelling process of theheart. Authors found that all FXIII−/− and FXIII−/+ mice died within 5days after MI due to left ventricular rupture. On the contrary, FXIII−/−mice that received five days of intravenous FXIII replacement therapyhad normal survival rates; though, their cardiac MRI demonstrated worseleft ventricular remodelling. Again, by using a FXIII-sensitivesensitive molecular imaging they found significantly greater FXIIIactivity in wild-type mice and in reconstituted FXIII−/− mice than innon-reconstituted FXIII−/− mice. Contextually, neutrophil cell migrationinto the MI scar was diminished in FXIII−/− mice but not inreconstituted FXIII−/− mice, and the physiological MMP-9 increasing,normally observed after MI, was 650% higher [4]. in non-reconstitutedFXIII−/− mice. This, together with the observation that collagen-1 levelwas 53% lower in FXIII−/− mice, demonstrate an imbalance in ECM turnoverand provides a possible explanations for the observed cardiac rupture in100% of the FXIII deficient mice. In confirmation of the latter, mice KOfor MMP-9 gene were protected against cardiac rupture and they survived.Thus, prevention of unrestrained MMP-9 upregulation by FXIII efficientlycontributes to protection against post-MI cardiac rupture.

After the publication of Nahrendorf [4], several other papers reporteddata in favour of the extraordinary role of FXIII in the post-MI healingfate. Most of them dealt with poor healing and adverse remodelling asthe primary cause of heart failure [5,6]. Other suggested FXIII as “Thecement of the heart after myocardial infarction” [7], able to contrastinfarct expansion and suggesting FXIII as a useful replacement therapyin patients with ventricular rupture [5].

The direct pro-healing properties of FXIII in post-infarcted heart (i.e.angiogenesis and cell proliferation/differentiation) and those mediatedby the FXIII-stabilized matrix into the wound (i.e. modulation of fibrinand new collagen synthesis/deposition) prompted researchers even topropose intra-myocardial injections of polymeric biomaterial componentsor utilization of fibrin-cell-seeded scaffold in the first phases of MI.This could help in reducing infarct size and facilitating stem cell orgrowth factors delivery improving in turn better heart healing andhigher survival [8,9].

Finally, the recently recognized role of the cellular immune response asessential in myocardial healing, ascribe to neutrophils, andmonocytes/macrophages key functions in post-MI healing [10-13]. Briefly,in the first hours after ischemia, neutrophils accumulate in theinfarcted myocardium and peak about within 24 hours; thereafter,monocytes/macrophages invade the lesion. Divergent and complementaryfunctions have been associated to different monocyte subsets during thecomplex healing phases. In the inflammatory phase, neutrophils, andmonocytes migrate in the infarcted myocardium to remove dead cells anddebris and promote ECM degradation by activated MMPs; subsequently,monocytes/macrophages produce cytokines to repress inflammatory signalsand regulate the formation of granulation tissue, essential for theproliferative phase. Now, new blood vessels, fibroblast proliferation,and ECM formation, favourite the maturation phase in which this tissueis substituted by a mature collagen based scar.

FXIII takes part to all of these functions, being at the intersection ofcoagulation, inflammation as well as wound healing [3]. In fact,recruitment of both macrophages and neutrophils are reduced in theinfarcted heart of FXIII−/− KO mice as well as phagocytic activity.FXIII is also contained in these particular inflammatory cells, and itbasically furnishes a more robust tri-dimensional meshwork withaugmented elastic and extensible properties. This facilitates new vesselformation and cell differentiation avoiding imbalance in ECM turnoverand attenuating inflammatory response to injury.

All the data described above are in favour that having appropriatelevels of FXIII at the injury site is essential requisite for optimalwound healing particularly in the earliest phases. Accordingly, recentreports strongly suggest the needing to explore new treatment strategiesto repair the injured heart, by augmentation of intrinsic wound healingthat occurs during the first 1 to 2 weeks after MI. This also because onone hand the existence of efficient acute care (angioplasty,thrombolyses) have reduced drastically acute infarct mortality, and onthe other the inadequate options to treat the increased number ofinfarct survivors has contributed to the growth of post-MI chroniccomplications in particular heart failure [1].

Accordingly, other studies suggest that a prompt FXIII supplementationcould help heart in healing itself, because of the lack/deficiency ofthis enzyme invariably leads to the worst-prognosis of healing aftermyocardial injury [5,6,14]. Although intervening at this step isconsidered a promising, but underexploited, useful time window betweenacute reperfusion efforts and therapy against anomalous cardiacremodelling/chronic heart failure, one must be cautious and keep in mindthat FXIII stabilizes blood clots (thrombus) which presence incoronaries is just responsible for occlusion/infarction [4].

The quality of infarct healing shortly after myocardial injury marks thefate of the patient for years to come. Nowadays, there no existdedicated laboratory tests to efficiently predict post-MI healingoutcome and/or to have prognostic information to help clinicians inearlier applying personalized treatment to avert anomalous ventricularremodelling and heart failure or other major adverse cardiac events(MACE).

OBJECT AND SUMMARY OF THE INVENTION

Object of the represent invention is to provide a novel biomarker fordetermining prognosis of a patient who suffered a myocardial infarction.

According to the invention, the above object is achieved thanks to thecompositions specified in the ensuing claims, which are understood asforming an integral part of the present description.

In an embodiment, the present disclosure provides for a diagnostic kitfor evaluating FXIII levels in a myocardial infarcted patient, havingFXIII levels prognostic value about recovery of the patient, wherein thekit comprises means for detecting FXIII levels in biological samples ofsaid patient, and at least four sample collecting tubes suitable tocollect at least four patient biological samples taken on the day ofmyocardial infarction (t0) and at least on the following three days (t1to t3).

A further embodiment of the instant disclosure concerns a diagnosticmethod for determining prognosis of a myocardial infarcted patient,wherein the levels of FXIII protein are determined on the day ofmyocardial infarction (t0) and at least on the following three days (t1to t3), wherein a lowering of FXIII level on any one of t0 to t3 below athreshold value is indicative of a poor prognosis.

The present disclosure provides the evidence that FXIII level monitoringin the very early phases of myocardial infarction helps the cliniciansin predicting infarction evolution with particular interest in thepossible post-myocardial infarction major adverse cardiac eventsestablishment and consequently determining therapeutic treatments.

BRIEF DESCRIPTION OF THE ANNEXED DRAWINGS

Further features and advantages of the invention will become apparentfrom the detailed description which follows and which is given purely byway of non limiting examples with reference to the annexed figures ofdrawings. These drawings are essentially in the form of diagrams showingexemplary delivery profiles of various active agents that may beachieved using compositions as described herein. Specifically:

FIG. 1: Mean and Median values of FXIII levels at the scheduled time inthe whole cohort of MI patients analyzed.

FIG. 2: Comparison of Mean and Median values of FXIII levels at thescheduled time in those cases who experienced during the follow-up anykind of MACE (right) versus those who didn't have MACEs (left).

FIG. 3: Comparisons of Mean and Median values of FXIII levels at thescheduled time between patients who experienced AMI (re-infarction) asfirst end-point in the follow-up after the first acute MI (right) versusthe remaining MACE subgroup (left).

FIG. 4: Mean and Median values of FXIII levels at the scheduled time inthose patients who experienced heart failure (HF) and in those whodeceased during the follow-up. In the dead subgroup (right), t30 was notreported due to the partial and scanty availability of samples (seeresults).

FIG. 5: Mean and Median values of FXIII levels at the scheduled time inthose patients who experienced unstable angina as main symptom duringthe follow-up.

FIG. 6: ROC curve of FXIII at day 4th (t4*); the associated informativedetails are reported in Table 1.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, numerous specific details are given toprovide a thorough understanding of embodiments. The embodiments can bepracticed without one or more of the specific details, or with othermethods, components, materials, etc. In other instances, well-knownstructures, materials, or operations are not shown or described indetail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the appearances of the phrases “in oneembodiment” or “in an embodiment” in various places throughout thisspecification are not necessarily all referring to the same embodiment.Furthermore, the particular features, structures, or characteristics maybe combined in any suitable manner in one or more embodiments.

The headings provided herein are for convenience only and do notinterpret the scope or meaning of the embodiments.

The present disclosure concerns the identification of a novel biomarkeruseful in determining potential recovery of patients who suffered amyocardial infarct.

In an embodiment, the present description provides for a diagnostic kitfor evaluating FXIII amounts in a myocardial infarcted patient, havingFXIII amounts prognostic value about recovery of the patient, whereinthe kit comprises means for detecting the amount of FXIII protein in atleast four, preferably six, biological samples and at least four,preferably six, sample collecting tubes suitable to collect the at leastfour, preferably six, patient biological samples taken on the day ofmyocardial infarction (t0) and at least on the following three to fivedays (t1 to t3 or t1 to t5).

A still further embodiment of the instant disclosure concerns adiagnostic method for determining prognosis of a myocardial infarctedpatient, wherein the amounts of FXIII protein are determined on the dayof myocardial infarction (t0) and at least on the following three days(t1 to t3), wherein a lowering of FXIII amount on any one of t0 to t3below a threshold value is indicative of a poor prognosis.

In a further embodiment, the threshold value of FXIII protein amount isevaluated as a percentage of a FXIII protein reference valueconventionally fixed as 100% (if not differently reported) of areference sample (normal control), wherein such a reference sample isobtained by standard reference materials like for example referencecalibration lyophilized pooled plasma.

As an example, the threshold value (or cut-off value), below which thepatient is at risk of a poor prognosis (i.e. heart failure or death),varies according to the time considered (t0-t5). All the thresholds areexpressed as percentage of FXIII protein amount of the normal control(reference). The threshold values for t0 to t5 (determined by the ROCanalysis), provided appreciable and significant results, i.e. allowed tocorrectly determine the prognosis of an infarcted patient. Accordingly,in the example below, patients who developed heart failure or those whodied after AMI are joined together because of their behaviours (in FXIIIconsumption) are very similar. Hence, comparing by the ROC procedure theFXIII levels of this MACE+ subgroup with the FXIII levels of all theMACE− group, the results were statistically significant.

In table 1 the threshold values of FXIII protein amount on any of t0 tot5 are provided.

TABLE 1 FXIII % Sensitivity/ Cut-off Specificity Time (ROC) (%) P AUC CI(95%) t0 88.0 69.23/70.43 <0.0001 0.720 0.663-0.769 t1 87.7 65.45/66.67<0.0001 0.700 0.638-0.754 t2 85.8 65.31/67.05 <0.0001 0.710 0.644-0.768t3 75.6 64.44/75.94 <0.0001 0.760 0.709-0.800  t4* 74.9 74.29/67.01<0.0001 0.740 0.677-0.787 t5 63.0 73.33/76.00 <0.0001 0.770 0.660-0.855ROC = Receiver Operating Characteristic; AUC = Area Under Curve; CI(95%) = Confidence Interval (95%). The reported thresholds were obtainedcomparing the FXIII levels of the MACE+ (heart failure and death) withthe whole MACE− subgroup.

The content of Table 1 indicates that the prognostic value of FXIII isalready informative in the earliest times (t0-t3). Anyhow, themonitoring of FXIII in a quite complete way (t0-t5) is recommended,because it is difficult to exactly predict the degree of FXIII proteinconsumption for any infarcted patient. As an example, the ROC curve atday 4 (t4; FXIII cut-off=74.9%) is provided in FIG. 6.

Today, FXIII assays are performed to determine the amount and/oractivity of this enzyme in blood to understand if the enzyme level maybe responsible or not for a determined pathological/clinical condition,generally haemorrhage. The test, if performed in “acute” condition (i.e.haemorrhage), must always be confirmed in a steady-state-condition farfrom the acute event. The assay is performed as an individual singletest, that is sufficient for a definite diagnosis.

Conversely, the instant disclosure provides data about the prognosticvalue of FXIII levels in post-infarcted patient: monitoring in acutephase the FXIII levels on a time window of four, preferably six, days(i.e. on the day of acute myocardial infarct (t0) and on the followingthree to five days (t1 to t3 or t0 to t5)) allows to predict the healingpotential (the degree to heal) of such a patient, regardless otherassociated and/or concomitant interfering/confounding factors.

The experimental data herein collected provide evidence that the acuteFXIII fall, due to heart injury, virtually happens in quite allmyocardial infarction patients but at different extent. This is causedby both coronary thrombus formation and tissue healing/myocardial scarformation. In detail, the present inventor found that a patientundergoing excessive FXIII consuming at the time of myocardial infarct(MI) is more prone to develop severe heart failure or death, whereasless drastic consuming is found in patients relapsing with re-infarctionor u-angina.

Having low/not optimal FXIII levels during the critical scar formationphase hampers heart healing giving back not properly tissue repair withchronic future consequences on myocardial performances. In thisrestricted period of time, the quality of infarct healing determines thefate of the patient for years to come.

Monitoring of FXIII levels during the acute MI enables clinicians toearly discover those patients at risk of poor post-MI prognosis givingthe possibility to intervene with targeted treatments.

Additionally, FXIII monitoring allows to recognize in advance thosepatients really in need to receive possible FXIII infusion therapy (toameliorate myocardial healing); indiscriminate FXIII infusion is, infact, responsible of undesirable pro-clotting function before reliablecoronary reperfusion after infarction is completely re-established andmust be performed only in patients with actual need thereof.

Taken together, these observations indicate FXIII a useful prognosticbiomarker with potential prospective in therapeutic handling of apatient after MI.

The present invention describes how different degrees in the fall ofFXIII levels allow to predict the risk to experience the major post-MIadverse cardiovascular events.

So monitoring the amount of FXIII drop observed in the acute MI phasesallows prognosticating whether such a patient is at risk to developfuture accidents.

The need to have an effective prognostic test is mainly due to the factthat starting from the seventies, large part of efforts in thecardiovascular field have been addressed to diagnostic and surgical(intervention) procedures reaching exceptional results. Due to theseeffective actions, the rate of patients who survived MI drasticallyimproved and they continue to rise.

The present invention concerns a test able to recognize in advance thosepatients at increased risk to develop additional cardiovascularaccidents in order to approach them with a tailor-made therapeuticaltreatment.

Nowadays, does not exist a prognostic test or a specific treatment toeffectively avert the adverse events secondarily to MI; there exists, infact, a restricted period of time (1-2 weeks from the establishment ofMI) during which it is possible to act in order to potentiate theintrinsic healing power of the heart and thus saving patients fromdeath.

For these reasons, the present disclosure concerns the assessment andmonitoring of FXIII levels in the routine laboratory-test-profile foracute myocardial infarction (AMI) detection. The classical marker testsfor myocardial infarction are merely passive indicators of heart damage,though very precocious and informative diagnostic tools. Conversely, thecomplex combined test evaluation herein disclosed results in preciousprognostic information very useful for the clinicians. Once verified thediminished FXIII levels it could be possible to act by adjunctive (alsotherapeutic) treatments. Assessment and monitoring of FXIII levels canalso be combined with the classical ischemic markers.

Materials and Methods

Patients and Methods

350 acute MI patients (whole group; mean age 68.2±12.95 years; 72.8%men) admitted to the Emergency Department of the University-Hospital ofFerrara were recruited.

Inclusion criteria were: prolonged chest pain occurring at restaccompanied by electrocardiography (ECG) ischemic changes. CK-MB and/orTroponin-I values were greater than the upper reference limit in twoseparate blood samples.

All noteworthy patient characteristic are reported in Table 2.

TABLE 2 Total STEMI NSTEMI (n = 350) (n = 252) (n = 98) P Age (y, SD,68.2 ± 12.95 67.1 ± 13.50 71.05 ± 11.33 <0.05 range) (31-80) (31-80)(38-80) Male (n, %) 255 (72.8) 186 (73.8)  69 (70.4) NS PCI (n, %) 316(90.3) 235 (93.25) 81 (82.6) <0.05 PCI = percutaneous coronaryintervention; STEMI = patients showing ST-segment elevation MI atenrolment; NSTEMI = patients NOT showing ST-segment elevation MI atenrolment.

Blood Samples

Blood was collected in Trisodium Citrate Coagulation tubes at admission(t0) and every 24 h for additional five days (t1-t5) from the acute MIevent. Control samples were drawn at least after 30-days (t30) to havebasal FXIII levels far from the acute ischemic events.

To exclude possible further in vitro enzyme degradation/activationadditional comparative samples were drawn in EDTA plus Aprotinin tubes.

Plasma was obtained by blood centrifugation at 2500 g×10 min at roomtemperature, and different aliquots were frozen at −80° C.

In order to verify, in actual fact, how different collection tubes couldinfluence the results, additional MI patients (n=45) were recruited andblood was drawn in duplicate by means of both the classical TrisodiumCitrate Coagulation tubes and the EDTA plus Aprotinin tubes. Therecruitment scheme was the same as above described (t0-t5, t30) and theplasma obtained was frozen (−80° C.) till the assessment.

FXIII Level Measurements

FXIII antigen levels were assessed by means of a Latex Reagent which isa suspension of uniform size polystyrene latex particles coated withrabbit polyclonal antibodies, highly specific for the A-subunit of FXIIIaccording to the manufacturer's instructions (InstrumentationLaboratory, Milan, Italy). Briefly the method is based on the principlethat when a sample (plasma) containing the A-subunit of FXIII is mixedwith the Latex reagent included in the kit, the coated latex particlesagglutinate. The degree of agglutination is directly proportional to theconcentration of FXIII antigen in the sample and is determined bymeasuring the decrease of transmitted light caused by the aggregates.The test is easy-to-perform, very fast, and routinely practicable.

Follow-Up (FU) and Statistics

The primary endpoint was a composite of cardiovascular death,re-infarction, heart failure or unstable angina (u-angina) at one-year.Major adverse cardiac events (MACEs) were retrospectively analyzed assingle variable or combined. Continuous data were presented as means±SD,with the significance of differences judged by t-test. Categoricalvariables were summarized in terms of number and percentages with thesignificance of differences judged by Chi-Square test. The recognitionof the FXIII threshold(s) at any period of time considered (t0-t5) wasobtained by means of the Receiver Operating Characteristic (ROC)analysis. Probability was considered significant at a level of P<0.05.

Results

FXIII was tested at the recruitment (t0) and every 24 h for additionalfive days (t1-t5) from the acute MI event. Control samples were drawn atleast after 30-days (t30) to have basal FXIII levels far from the acuteischemic event.

Table 3 shows the results of FXIII monitoring in acute MI patientsobtained using standard sample collecting tubes (indicated TrisodiumCitrate Coagulation tubes) and sample collecting tubes containing atleast one anti-proteolytic agent and at least one anti-coagulating agent(indicated EDTA plus Aprotinin tubes).

Though slightly lower values resulted from the EDTA plus Aprotinintubes, no significant differences were obtained by comparing the twodifferent vials in which blood was collected. Anyhow, making use oftubes containing at least one anti-proteolytic agent and at least oneanti-coagulating agent is recommended in those situations in which a notso rapid assessment of the test can be possible (delay ≧4 hours from thetime in which blood is taken).

The anti-proteolytic agent can be selected among aprotinin, heparin,antithrombin, hirudin, EDTA (Ethylenediaminetetraacetic acid), EGTA(Ethylene glycol-bis(β-aminoethyl ether)tetraacetic acid), Leupeptin,iodoacetamide, APMSF ((4-Amidino-phenyl)methane-sulfonyl fluoride),4-(2-Aminoethyl)-benzenesulfonyl-flouride hydrochloride,a2-Macroglobulin.

The use of collecting tubes containing at least one anti-proteolyticagent allows, in fact, to determine the actual FXIII protein amount inthe infarcted patient avoiding further consumption of FXIII proteincontained in the collecting tube because of the presence of theanti-proteolytic(s) completely blocks any further (in vitro) enzymeactivation. This precaution warrants a more reliable FXIII detection,better reflecting that particular in vivo status.

TABLE 3 t0 t1 t2 t3 t4 t5 t30 Trisodium Citrate Coagulation tubes Mean99.48 97.68 94.72 90.72 85.24 76.51 110.15 Median 98.4 95.30 94.20 88.7083.20 74.05 108.20 SD 30.57 28.06 29.12 30.30 24.93 27.02 20.34 min40.60 39.90 32.80 29.5 20.90 20.00 60.00 max 186.40 181.10 179.80 169.10136.20 138.25 147.90 EDTA plus Aprotinin tubes Mean 98.34 96.87 93.6189.16 83.08 75.45 107.34 Median 94.40 95.15 92.30 87.90 72.65 74.00105.90 SD 31.87 32.34 34.40 31.25 28.72 29.19 27.37 Min 41.05 38.7534.70 30.5 22.05 21.90 61.25 Max 193.90 190.90 182.50 178.20 139.60135.25 152.70 P 0.92 0.32 0.30 0.35 0.28 0.84 0.80

FIG. 1 shows the mean and median levels of FXIII during the assessmentperiod in the whole cohort.

FIGS. 2 to 5 show the FXIII levels in different subgroups of patientsduring the six days (t0-t5) and at the final check (t30).

Globally, a significant FXIII level drop was clearly observed at t4-t5as both mean and median regardless the patients experienced or not majoradverse cardiac events (MACE) (FIGS. 2B and 2A, respectively).

Interestingly, the combined endpoint analysis showed that the FXIII fallwas significantly stronger considering those patients who experiencedmajor adverse cardiac events (MACE) (MACE+; n=101; 28.8%) when comparedwith the rest of patients free of major post-MI events (MACE−; n=249;71.1%).

It's noteworthy the observation that MACE+ patients presented withsignificantly lower FXIII levels at t0 and at any time considered asboth mean and median. The data of FXIII levels in the t0 to t5 timewindow for MACE− and MACE+ patients are provided in Tables 4 and 5,respectively.

TABLE 4 MACE− (n = 249) t0 t1 t2 t3 t4 t5 t30 Mean 103.73 102.10 101.3198.02 89.34 83.37 109.53 Median 102.20 100.53 97.80 94.05 85.20 91.00108.35 SD 28.77 29.17 31.55 29.74 25.58 23.53 24.95 min 46.30 42.6042.40 40.60 38.30 37.00 54.80 max 236.80 201.20 198.90 193.90 149.60136.60 172.70

TABLE 5 MACE+ (n = 101) t0 t1 t2 t3 t4 t5 t30 Mean 88.69 86.22 82.6876.28 71.00 65.07 91.94 Median 81.00 81.00 78.00 72.00 66.00 60.55 88.95SD 29.41 29.87 29.71 29.10 29.01 31.12 24.28 min 35 32.8 31.5 30.5 20.918 54 max 167.2 160.5 158.4 151 148 136.6 159

Interestingly, the degree of the FXIII lowering became even stronger inthe MACE+ subgroup (FIG. 3A) when those who experienced acute myocardialinfarction (AMI) as first end-point in the follow-up were excluded.Indeed, in these latter (AMI), the FXIII drop mean behaviour stronglydiverged from the other groups considered, being apparently not affectedby a sharp fall of FXIII (FIG. 3B).

Conversely to what happened in the previous described patients (heartfailure or dead subgroups), here those who developed AMI, diverge fromthe other patients being paradoxically less “FXIII-consuming” even thanthe MACE− group. For this reason we did not compute by ROC analyses thiskind of results, but strongly underline their importance suggesting theyshould be taken in consideration and defer their final interpretation.

To note, these patients (AMI) presented with higher mean FXIII level att0, and never went below a mean value of 94.0% (t4).

According to the literature on the role of FXIII in recovering of tissuedamages, that is low FXIII levels might be responsible for anomalouspost-MI heart/wall remodelling causing heart failure or even death (dueto inefficient/poor myocardial scar formation at the injury site), thepresent inventor specifically analyzed how FXIII levels were in thosepatients who experienced post-MI heart failure (HF) or death.

HF patients showed awful FXIII mean reduction and their trend wascharacterized by a sharper slope. Though they reached very low meanFXIII levels (FIG. 4A), they showed appreciable and normal recovery ofFXIII at t30. Conversely, those patients who died due to cardiovascularcomplications related to the acute experienced MI (FIG. 4B), presentedat t0 with slightly lower levels and reached t4-5 with mean and medianFXIII values resembling those of the HF subgroup. Unfortunately, t30 wasnot collected because of large part of these patients died before/oraround that time or were definitely not available due to concomitanttreatments or complications.

Finally, those patients who experienced u-angina as the prevailingsymptom during the follow-up (FIG. 5), though they showed a slow rate inFXIII decreasing, did not show at t30 the classical recover of FXIIIlevels. This could mean they “continue” to spend/consume FXIII also farfrom the acute infarction event, possibly due to the chronic relapsingin angina attacks.

Similarly to what we did in the previous less “FXIII-consuming” AMIpatients, also data from anginous cases were not computed by ROCanalyses. We strongly underline as well, their importance suggestingthey should be taken into consideration and defer their finalinterpretation.

Summarizing, the test/procedure presented here gives very informativeresults as a prognostic indicator of those MACEs related to excessiveFXIII consumption (HF or death). Conversely, though noteworthy, the“low” FXIII consumption observed in anginous and AMI patients deservefuture and definite investigations, ascribing anyhow to them a strongpotential clinical relevance.

REFERENCES

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1. Diagnostic kit for determining FXIII protein levels in biologicalsamples of a myocardial infarcted patient, the kit comprising: means fordetecting the amount of FXIII protein in the biological samples, and atleast four sample collecting tubes suitable to collect the at least fourpatient biological samples taken on the day of myocardial infarction(t0) and at least on the following three days (t1 to t3).
 2. Diagnostickit according to claim 1, wherein the kit comprises at least two furthersample collecting tubes suitable to collect at least two further patientbiological samples taken on at least the fourth and fifth day followingmyocardial infarction (t4 to t5).
 3. Diagnostic kit according to claim1, wherein the sample collecting tubes contain each at least oneanti-proteolytic agent and an anti-coagulating agent.
 4. Diagnostic kitaccording to claim 3, wherein the at least one anti-proteolytic agent isselected among aprotinin, heparin, antithrombin, hirudin, EDTA(Ethylenediaminetetraacetic acid), EGTA (Ethyleneglycol-bis(β-aminoethyl ether)tetraacetic acid), Leupeptin,iodoacetamide, APMSF ((4-Amidino-phenyl)methane-sulfonyl fluoride),4-(2-Aminoethyl)-benzenesulfonyl-flouride hydrochloride,a2-Macroglobulin.
 5. Diagnostic kit according to claim 1, wherein themeans for detecting the amount of FXIII protein comprises a specificbinding ligand of FXIII protein, the specific binding ligand of FXIIIprotein being preferably coated onto a solid phase.
 6. Diagnostic kitaccording to claim 5, wherein the specific binding ligand is selectedbetween anti-FXIII polyclonal and monoclonal antibody.
 7. Diagnostic kitaccording to claim 1, wherein the kit comprises at least one normalcontrol, wherein the normal control comprises a fixed amount of FXIIIprotein.
 8. Method for predicting prognosis of a myocardial infarctedpatient, the method comprising: providing at least four biologicalsamples of the infarcted patient, the at least four samples beingcollected on the day of myocardial infarction (t0) and at least on thefollowing three days (t1 to t3); determining the amounts of FXIIIprotein in each biological sample; wherein a lowering of FXIII proteinamount in any one of t0 to t3 biological samples below a threshold valueis indicative of a poor prognosis.
 9. Method according to claim 8,wherein the method further comprises: providing at least two furtherbiological samples of the infarcted patient, the at least two furthersamples being collected on at least the fourth and fifth day followingmyocardial infarction (t4 to t5); determining the amounts of FXIIIprotein in t4 and t5 biological sample; wherein a lowering of FXIIIprotein amount in any one of t4 and t5 biological samples below athreshold value is indicative of a poor prognosis.
 10. Method accordingto claim 8, wherein the threshold value of FXIII protein amount isevaluated as a percentage of the FXIII amount of a positive control. 11.Method according to claim 8, wherein the threshold value of FXIIIprotein amount for each t0 to t5 biological sample is expressed aspercentage of FXIII protein amount of a normal control, such amountbeing fixed to 100%, wherein the threshold values are: Threshold sample(FXIII %) t0 88.0 t1 87.7 t2 85.8 t3 75.6 t4 74.9 t5 63.0


12. Method according to claim 8, wherein the patient biological samplesare collected in sample collecting tubes containing at least oneanti-proteolytic agent and an anti-coagulating agent.
 13. Methodaccording to claim 8, wherein the patient biological samples are wholeblood, plasma or serum samples.